Carbon Metabolism Molecular Mechanisms Of D-fructose Promotion On The Furanone Production In Zygosaccharomyces Rouxii

Zygosaccharomyces rouxii(Z.rouxii)is a kind of important yeast in soy sauce and soy sauce fermentation food.It can metabolize sugars into organic acids,alcohols and other flavor metabolites and also produce aromatic compounds.Among of them,4-hydroxy-2,5-dimethyl-3(2H)-furanone(HDMF)and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone(HEMF)are the main aroma components in soy sauce and soy sauce,but their metabolism mechanism is not clear in Z.rouxii.It was found that the addition of D-fructose could increase the furanone production in Z.rouxii.Therefore,the carbon metabolism pathway of exogenous D-fructose regulating the synthesis of HDMF and HEMF by the yeast would be helpful to provide theoretical basis for the creation of fragrant compound cell factory in the biosynthesis of food by the yeast.In this paper,we studied the gene expressions of HDMF and HEMF in various fermentation stages,and enzymology method was used to conduct joint demonstration at the protein level.It provides a new theoretical basis for the biosynthesis of furanones.Transcriptomics and metabolomics were combined to further reveal the metabolic mechanism from the molecular perspective.The study focused on the following aspects:(1)Illumina Hi Seq TM high-throughput sequencing platform was performed to check the D-Fructose-associated in YPD culture of Z.rouxii transcriptomes.Clean reads were obtained at different fermentation stages.HTSeq software was used to analyze the gene expression level of each sample,and hmmscan was used to obtain the new gene GO annotation file.Through KEGG analysis,it was found that there were complete glycolysis(EMP),pentose phosphate pathway(PP pathway)and carbon metabolism pathway of tricarboxylic acid cycle(TCA)in the yeast.FBA and 6PGDH were key genes that respectively regulated the synthesis of furanone precursors dihydroxyacetone phosphate(DHAP)and ribulose-5-phosphate.(2)The growth characteristics and the change of furanone contents were evaluated under D-fructos addition in Z.rouxii.It was found that the biomass in D-fructose involved YPD was 2.5-folds of that without D-fructose,and the growth state of the colony was better.The addition of exogenous D-fructose significantly increased the production of HDMF and HEMF in the yeast,which reached 98 mg/L and 25 mg/L at 5 d,and increased the capacity of acid and ketone production.Those results showed that D-fructose was more beneficial to the growth of Z.rouxii and the production of flavor substances.(3)In order to investigate the effects of exogenous D-fructose on the transcription of furanone,real-time quantitative PCR was used to detect the changes of gene expression related to furanone precursor in EMP and PP pathways.The results indicated that the expression of HK,PFK1,G6 PI,FBA,TPI,6GPL,TKT and 6PGDH were positively related to furanone synthesis with significant difference,and played positive regulatory roles.It is suggested that these genes may promote the synthesis of furanones during the process of enzymatic catalysis and spontaneous chemical transformation.(4)GC-MS method was used to detect the changes of metabolites related to furanone synthesis.It was found that the metabolites related to furanone synthesis,such as fructose-1.6 bisphosphate and ribulose-5-phosphate,were increased and the yield and the organic acid and esters flavor metabolites were also increased.Furthermore,the metabolic levels showed that exogenous D-fructose could improve the biosynthesis of furanone.The structure of FBA gene was analyzed by bioinformatics.The total length of FBA gene was 1086 bp,encoded 361 amino acids.It was a hydrophilic protein and belongs to non transmembrane protein.?-helix and irregular curl were the main structural elements of FBA protein.